Paper II of IV in my doctoral thesis:

Characterization of an Experimental Vaccine for Bovine Respiratory Syncytial Virus.

Sara Hägglund, Kefei Hu, Krister Blodörn, Boby Makabi-Panzu, Anne-Laure Gaillard, Karin Ellencrona, Didier Chevret, Lars Hellman, Karin Lövgren Bengtsson, Sabine Riffault, Geraldine Taylor, Jean-Francois Valarcher, Jean-François Eléouët
Clinical and vaccine Immunology: CVI 05/2014; 21(7). DOI:10.1128/CVI.00162-14


Bovine respiratory syncytial virus (BRSV) and human respiratory syncytial virus (HRSV) are major causes of respiratory disease in calves and children, respectively, and are priorities for vaccine development. We previously demonstrated that an experimental vaccine, BRSV-immunostimulating complex (ISCOM), is effective in calves with maternal antibodies. The present study focuses on the antigenic characterization of this vaccine for the design of new-generation subunit vaccines. The results of our study confirmed the presence of membrane glycoprotein (G), fusion glycoprotein (F), and nucleoprotein (N) proteins in the ISCOMs, and this knowledge was extended by the identification of matrix (M), M2-1, phosphoprotein (P), small hydrophobic protein (SH) and of cellular membrane proteins, such as the integrins αVβ1, αVβ3, and α3β1. The quantity of the major protein F was 4- to 5-fold greater than that of N (∼77 μg versus ∼17 μg/calf dose), whereas G, M, M2-1, P, and SH were likely present in smaller amounts. The polymerase (L), M2-2, nonstructural 1 (NS1), and NS2 proteins were not detected, suggesting that they are not essential for protection. Sera from the BRSV-ISCOM-immunized calves contained high titers of IgG antibody specific for F, G, N, and SH. Antibody responses against M and P were not detected; however, this does not exclude their role in protective T-cell responses. The absence of immunopathological effects of the cellular proteins, such as integrins, needs to be further confirmed, and their possible contribution to adjuvant functions requires elucidation. This work suggests that a combination of several surface and internal proteins should be included in subunit RSV vaccines and identifies absent proteins as potential candidates for differentiating infected from vaccinated animals.

Excerpt from my doctoral defense presentation

Classic BRSV-ISCOMs evaluated in study I induced exceptional protection in calves with BRSV-specific maternally derived antibodies (MDA). These BRSV-ISCOMs were produced using purified antigen from lysate of BRSV-infected cells and in this purification process only one fraction of the total antigen is used in the final vaccine. This leads to the question: What’s in the BRSV-ISCOMs?

To answer this question, and to facilitate standardization of the formulation, but also enable the design of subunit vaccines, the BRSV-ISCOMs were characterized in study II: by electron microscopy, and by direct and indirect detection of proteins. BRSV-ISCOMs used in study I contained 188 µg of proteins, and previous studies of BRSV- and HRSV-ISCOMs have identified the F and N, and the F and G proteins, respectively. This figure illustrates how we used SDS-PAGE and digital image analysis to identify BRSV proteins F and N, and to approximate their quantities in our BRSV-ISCOMs. Together, F and N constituted approximately half of the proteins in BRSV-ISCOMs, with 5 times more F than N. In addition, we identified BRSV-proteins G, M, M2-1, P and SH by mass-spectrometry and using monoclonal antibodies, but could not detect BRSV-proteins M2-1, NS1, NS2 or L. Cell derived proteins, predominantly integrins, were also identifies by mass spectrometry.


We also verified the presence of BRSV-proteins F, N, G and SH by detecting significant titers of protein specific antibodies in BRSV-ISCOM-immunized calves before challenge in study I. The blue bars represent mean titers of protein-specific antibodies in BRSV-ISCOM-immunized calves, whereas the arrows represent the mean titers of the control groups, as indicated in the legend.


To summarize study 2, we showed that proteins F and N were abundant in BRSV-ISCOMs, with 5 times more F than N, but also identified proteins G, M, M2-1, P and SH. We also showed that BRSV-ISCOMs induced antibodies against F, N, G and SH. These findings, which were published in CVI, suggest which proteins may be important to the protection observed in study I, and provided information that can be used to standardize the BRSV-ISCOM formulation, or that can be used to design new subunit vaccines.

In paper III, we refined and characterized a BRSV challenge model in calves and a set of tools to evaluate challenge outcomes, to facilitate further studies of vaccine candidate.